This invention relates to combinatorial chemistry, and more specifically to combinatorial libraries comprising solid supports in the form of support units (particles, beads and the like), each of which is labeled with one or more metals that provide a code for identifying the compounds that are or were attached to the bead.
In the field of combinatorial chemistry, libraries of chemical compounds are made for screening to determine which chemical compounds are active for a particular use, such as agonism or antagonism of a receptor. Usually this screening is carried out by performing assays on each member of the library or groups of members of the library. The compounds that have the desired activity as determined by the assay method are then made on a larger scale for more thorough testing.
Numerous strategies have been designed for testing and tracking the compounds being tested in these mass screenings so that the compounds that have activity in the assays can be readily identified after a positive assay. One of these strategies involves the synthesis of compounds (often referred to as ligands) on the solid support units such that each support unit carries a single compound. The compounds can then be assayed individually, either while they are still attached to the support unit, or more typically, after being cleaved from the support unit. Identification of the compound that is or was on the support unit after a positive assay result is still an ongoing source of difficulty. Since large numbers of support units are used (typically in the hundreds or thousands), the individual support units are not handled and tracked separately. For example, in a split pool synthesis, the support units are synthesized and manipulated in groups for each synthetic step and for assays. Even though each support unit may have only one kind of ligand bound to it, the individual support units are mixed with a large number of other support units, each having a different ligand bound to it. This kind of mass screening makes it impractical or impossible to keep track of the individual ligands as they are synthesized and assayed. As a result, after the assays have been completed, the ligands that are present on the beads that have the desired activity must still be identified. Either the ligand can be analyzed, as by mass spectrometry, or the ligand can be identified based on information contained in the support unit to which it is or was previously attached. To make analysis of the compound (the ligand) being assayed easier, schemes have been developed for encoding the support units by placing chemical markers or xe2x80x9ctagsxe2x80x9d on the support units and then using those tags to identify the chemical compound (ligand) that was originally synthesized on the support unit.
These chemical markers have taken at least two forms. In one, a unique sequencable oligomer, such as a polynucleotide or polypeptide oligomer, is synthesized in parallel with the compound that is being tested on the support unit. The nucleotide or peptide sequence is then determined for the units that have positive assays to determine the compound that has the desired activity in the assay. See for example, WO 93/06121; Brenner, et al., Proc. Natl. Acad. Sci. USA (1992), 89, 5381; Kerr, et al., J. Am. Chem. Soc (1993), 115, 2529; Lebl, Pept. Res. (1993), 6 (3), 161; and Lebl, Proc Natl. Acad. Sci. USA, (1996), 93, 8194. This approach to chemical coding requires the synthesis of a complete second, parallel library of oligomers that serve as chemical markers. This method can be very cumbersome and has the limitation that the syntheses of the oligomer/chemical marker and the molecules being tested must be compatible with one another.
A second approach to marking the support units involves attaching combinations of chemical markers to the support unit. In this approach, the information that identifies the support unit is carried in the combination of what markers are present and what markers are not present, and does not rely on the sequence of the markers. The chemical markers can each be attached directly to the support unit in some way or can be attached to each other and then attached as a group to the support unit. The information needed to identify the chemical compound that was synthesized on the support unit for testing is not retrieved by sequencing the markers, but rather is obtained by determining which markers are present and which markers are absent. This approach is inherently easier, since making and then later analyzing a molecular sequence is much more time consuming and difficult than just creating a code by attaching individual markers to a support and then later determining what markers are present without having to determine the order in which they are attached. Furthermore, only a few kinds of sequences can be determined using automated technology, such as polypeptides and polynucleotides.
The chemical markers can be used to provide a code, based on which chemical markers are present and which markers are absent. One very convenient and efficient kind of code is a binary code, where each chemical marker is represented as a digit in a binary number, with its presence or absence representing the two choices (i.e. xe2x80x9c1xe2x80x9d or xe2x80x9c0xe2x80x9d) for the binary digit. Examples of organic chemical markers that have been used in this approach include aryl ether carbenes, which are attached to the support unit at low levels compared with the molecule being synthesized during each step of a split pool synthesis, and are then decoded by cleavage of the aryl carbene residues from the support unit followed by gas chromatographic analysis. See for example, Still, et al. U.S. Pat. No. 5,563,324; Still et al., WO 94/08051; Still et al., WO 95/26640; Still et al., Proc. Natl. Acad. Sci. USA (1993), 90, 10922. Another example of a binary encoding scheme using organic markers is based on secondary amines assembled as N-amidomethyl polyglycines, Ni, et al, J. Med. Chem. (1996), 1601; Gallop, et al., U.S. Pat. No. 5,846,839.
Other methods for encoding support units use physical encoding, such as bar codes, as for example WO 97/15390. Radio frequency has also been utilized, as for example by IRORI, in Ang. Chem. Int. Ed. Engl. (1995), 34 (20), 2289; Ontogene, in J. Am. Chem. Soc. (1995), 117, 10787; and Mandecki, in WO 97/19958. Other marking methods include fluoroescence encoding, as for example in WO 95/32425 and Egner et al., Chem. Comm (1997), 735; and isotope ratio encoding, as for example in Geysen, et al., Chem. Biology (1997), 3 (8), 679; Geysen et al., WO 97/37953; Wagner et al, Combinatorial Chem. High Throughput Screening (1998), 1, 143; and Weinstock et al., WO 97/29371. These methods all have limited utility for general combinatorial libraries.
A variation on chemical encoding involves the use of metal ions rather than organic chemical residues to encode combinatorial libraries on solid supports. See Rink, et al., WO 96/30392, which reference is incorporated by reference into this application in its entirety. In this method, soluble salts (e.g. nitrates) of the lanthanide metal series are absorbed into the support units at each step of the split pool synthesis of the organic compounds (ligands) that are being attached to the support units. The metal salts in solution are added to the beads, which are suspended in the solvent.
A different metal salt or salts may be added to the support after each new step in the split pool synthesis. Analysis of the metal content of the support units and comparison with a key of what the various metals represent enables identification of the compounds.
Although the metal salts are not covalently attached to the support units, they are reported to remain in the beads throughout the synthesis in the form of soluble salts. The inventors report that sufficient quantities of the soluble metal salts xe2x80x9csurprisinglyxe2x80x9d remain in the beads through the subsequent reaction steps so that the presence or absence of the metal salts can be determined at the end of the process by methods that are customarily used in the analysis of elements or element ions, such as total reflection x-ray fluorescence spectrometry (TRXF), neutron activation followed by gamma spectrometry, or mass spectrometry, particularly inductively coupled plasma-mass spectrometry (ICP-MS).
However, the use of soluble metal salts can result in the loss of metal ions and/or crossover of metal ions between support units, making analysis difficult and/or uncertain. This can happen even under ideal conditions, and is more likely to happen if conditions are not ideal, as for example, if the solvent is heated, if the solvent contains or can act as a ligand for the metal ion, or if the metal salt is highly soluble in the solvent. Leaching and loss of the salts and crossover to other beads was observed with the salts that were used in the experiments disclosed herein. There is therefore a need to have support units and methods of preparing support units that are labeled with metal ions or other markers and that are stable to varied conditions, such as temperature, organic reagents, and solvents.
In the present invention, a solution of a soluble salt, the anion of which forms insoluble salts with the metal cations that are used to label (xe2x80x9cencodexe2x80x9d) the support units, is added to the labeled support units to prevent or decrease the loss of the metal used to label the support units. Loss of metal is believed to be prevented by the formation of insoluble or poorly soluble salts of the metals that are used to label the support units. Support units are the individual units of solid support that are used. Support units are generally beads, preferably porous beads.
For example, the support units can first be labeled with one or more metal salts in solution (e.g. AgNO3 in water). The metal-labeled support units can then be treated with a solution of Na2S, which is expected to form an insoluble salt of the metal that is used as a label (e.g. Ag2S). When this method is used, support units that are labeled with metal tags (salts) do not lose their metal labels as readily, if at all. The labeled support units are more stable to the harsh reaction conditions that are often needed for the synthesis or cleavage of the ligands, such as for example heating the support to 70xc2x0 C. in acetic acid overnight, without loss of the metal tags.
Another means of achieving improved retention of metal is to include linker groups that contain as part of their structure a moiety, such as sulfur, that forms non-covalent bonds to the labeling metals. A linker is one molecule or two or more molecules covalently bound together, where the linker connects the ligand to the functional groups of the support unit. Linkers generally have two reactive functional groups so that they can be connected to the support unit and to the ligand. Commonly used linkers include 4-hydroxymethylbenzoic acid (HMBA) and 4-hydroxymethylphenylacetic acid. These linkers are used because they are stable to cleavage under reaction conditions typically encountered in preparing a combinatorial library, but at the same time they are readily cleaved under a particular set of reaction conditions. A standard kind of linker molecule, such as HMBA, can be bound to a sulfur-containing molecule or molecules to form a linker that also includes a sulfur-containing moiety to achieve good bonding to the metals and also to achieve the properties typically desired from linkers.
When sulfur or another moiety that forms non-covalent bonds to the labeling metals is present in the linker molecules or elsewhere in the support units, the uptake and retention of the encoding metal salts by the resin is improved. For example, the carboxyl end of a tripeptide of methylated cystein can be attached to the amine groups of aminomethyl polystyrene to form a xe2x80x9cpre-sulfurized resin,xe2x80x9d where the pre-sulfurized resin is in the form of a bead. A hydroxymethylbenzoic acid (HMBA) molecule can then be attached to the free amine group of the tripeptide, and the sub-units of the ligand can be attached one sub-unit at a time in sequence to the hydroxy end of the HMBA to form the ligand on the support unit.
Use of beads or support units comprised of the pre-sulfurized resin encoded with metal cations gives better retention of the metal ion, minimizes cross contamination of the labeling metals between the support units, and results in a faster and more accurate determination of the code that is on the bead or support unit.
Furthermore, the combination of both of the above methods of stabilizing the metal on the support unit, i.e. by: (1) attaching sulfur-containing linker molecules to the support unit, and (2) treating the metal-labeled support units with a solution of an anion that forms poorly soluble or insoluble salts of the labeling metals, gives even better, more reliable retention of the labeling metals, and so far is the best way of using metal cations to encode the ligands of a combinatorial library.
This approach to encoding the support has the advantage of causing only minimal restrictions in the kinds of chemical reagents, solvents and conditions that can be used in the library synthesis, and also allows fast decoding without the necessity of cleaving the metal tags off of the solid support. Decoding of the metal is most conveniently done by mass spectral analysis, such as inductively coupled plasma mass spectrometry (ICP-MS). The preferred mass spectral process for decoding is a laser ablation-ICP-MS technique. This method requires very little time (less than 20 seconds) to decode each bead. The laser ablation equipment can be automated to scan large numbers of beads in arrays on plates. This method therefore offers the capability of decoding a whole library automatically, making it possible to obtain a detailed structure-activity relationship (SAR) from biological screening. Most other encoding-decoding methods previously used are not readily automated and are therefore only suitable for decoding a few active beads in a particular assay, with each new assay of a library sample requiring a different decoding step.
The present invention is a method of using and stabilizing metal salts to encode combinatorial support units in the preparation of combinatorial libraries. The invention is also a method of making encoded combinatorial libraries and of decoding combinatorial libraries to determine what ligands are on particular beads or have particular kinds of activity. The combinatorial support units and the libraries that are made using this methodology are all new.
A combinatorial library of this invention comprises a plurality of ligand-bearing support units, where the ligand-bearing support units comprise (a) a solid carrier, (b) one or more ligands covalently bound to the solid carrier, and (c) one or more encoding metal salts impregnated on the support unit, as follows:
(a) The carrier is the material that forms the support unit and contains functional groups to which the ligands are covalently bound, where the functional groups and the ligands are optionally connected by linker groups. The linker groups are organic residues that connect the carrier and the ligand and are covalently bound both to the solid carrier and to the ligand .
(b) The ligand is an organic compound covalently bound to the functional groups of the carrier or to the linker groups.
(c) The encoding metal salts include one or more encoding metal cations, which are distributed in their natural isotope abundance or in non-natural isotope abundances, where the distribution of the encoding metal cations provides a code for identifying the ligand or ligands that are attached to the support unit, and one or more anions that form insoluble or poorly soluble salts of the encoding metal cations in the solvent or solvents which are used to prepare the combinatorial library.
The metals that are used as labels can be used in their natural isotope abundances. Alternatively, other isotope abundances, which would be man-made (non-natural), can also be used. Generally, this would be a single isotope, which makes quantitative mass spectral measurement easier. The use of single isotopes also is advantageous in that it greatly increases the number of possible labels.
xe2x80x9cInsolublexe2x80x9d and xe2x80x9cpoorly solublexe2x80x9d conform with common usage. Salts that are insoluble or poorly soluble precipitate out of solution when solutions containing the cations and anions are mixed. The salts do not dissolve appreciably.
In the combinatorial library described above, the ligand may consist of two or more sub-units which are covalently bound to each other, and which are generally assembled sequentially on the support unit. The ligand is the organic compound whose chemical, biological, or other activity is being evaluated.
The encoding metal salts in this combinatorial library are impregnated onto the support unit by treating the support unit with a solution of a soluble salt of the encoding metal cations to form a support unit which is labelled with a soluble encoding metal salt, and then treating the support unit having the soluble encoding metal salt with a solution of a salt having an anion that forms insoluble or poorly soluble salts when combined with the encoding metal cations. This yields a stabilized encoding metal salt, wherein the salt has been stabilized against dissolution from the support unit.
In the combinatorial library described above, the solid carrier is generally a synthetic polymeric compound, and the support units are usually in the form of beads, particularly porous beads. The polymeric compound is normally polystyrene, optionally crosslinked with divinylbenzene, where the polystyrene includes functional groups that can react with other compounds. The ligands or the optional linker groups are bound to the functional groups of the polystyrene.
In preferred embodiments of the combinatorial library described above, the carrier and/or the optional linker groups includes one or more moieties that enhances the uptake and retention of the encoding metal salt. Preferably these moieties are in the linker groups. These moieties increase the initial loading of metal ions in the solid support and decrease the solubility of or rate of dissolution of the encoding metal salt by acting as a ligand that is non-covalently bound to the encoding metal cation.
In the combinatorial library, the encoding metal salts that are stabilized against dissolution include encoding metal cations which form soluble salts with one or more anions selected from the group consisting of nitrates, hydroxides, chlorides, acetates, and sulfates, and the encoding metal cations form insoluble or poorly soluble salts with one or more anionic groups selected from the group consisting of sulfides, sulfates, oxides, hydroxides, halides and carbonates. The encoding metal cations are usually selected from the group consisting of the transition metals, the lanthanides, the actinides, Sr, Ba, Tl, In, Sb, and Bi. Often, the encoding metal cations are selected from the group consisting of the Group VIIIB, IB and IIB transition metals. Preferred encoding metal cations include Pd, Ru, Rh, Pt, Ag, Ni, Cu, Co and Hg, including individual isotopes of each of these metals.
In many cases, the encoding metal salts are stabilized against dissolution by treatment with a solution of sulfide salts or precursors that can generate sulfide ions in situ.
This invention also is an improvement in combinatorial libraries that include a plurality of support units, wherein each support unit comprises (a) a solid carrier, (b) one or more ligands covalently bound to the solid carrier or to a linker group that is covalently bound to the solid carrier, and (c) one or more encoding metal salts impregnated on the support unit, where the encoding metal salts include encoding metal cations, and the distribution of the cations provides a code that identifies the ligand or ligands. The improvement is that the support unit also includes one or more anions that form insoluble or poorly soluble salts of the encoding metal cations in the solvent or solvents which are used to prepare the combinatorial library.
The combinatorial library is improved in that the encoding metal salts are impregnated onto the support unit by first treating the support unit with a solution of a soluble salt of the encoding metal cations to form a support unit labelled with a soluble encoding metal salt, and then treating the support unit including the soluble encoding metal salt with a solution of a salt that includes an anion that forms insoluble or poorly soluble salts when combined with the encoding metal cations, thereby stabilizing the encoding metal salt against dissolution from the support unit.
A further improvement is achieved when the support units include linker groups covalently bound to the solid carrier, where the linker groups include one or more moieties that enhances the uptake and retention of the encoding metal ions. An example would be a sulfur-containing linker group.
The invention furthermore is a method of preparing an encoded combinatorial library which includes a plurality of ligand-bearing support units, wherein the ligand-bearing support units include (a) a solid carrier, (b) one or more ligands covalently bound to the solid carrier, and (c) a plurality of encoding metal salts impregnated on the support unit, the metal salts providing a code for identifying the ligand. The method includes the steps of:
(1) providing support units which include a solid carrier having functional groups, the functional groups being optionally connected to linker groups, the linker groups being organic residues covalently bound to the functional groups of the solid carrier and having functional groups for covalent binding to the ligand;
(2) covalently attaching a ligand or a first sub-unit of a ligand that will have more than one sub-unit to the functional group of the carrier or to the functional group of an optional linker group, in which case the sub-unit has a functional group for covalent binding to a second sub-unit; and
(3) impregnating the support unit with one or more encoding metal salts, the salts being composed of one or more encoding metal cations, which are distributed in their natural isotope abundance or in a non-natural isotope abundance, where the encoding metal cations provide a code for identifying the ligand or sub-unit that is attached to the support unit, and the encoding metal salts are impregnated onto the support unit by treating the support unit with a solution of a soluble salt of the encoding metal cations to form a support unit labeled with the soluble encoding metal salts, and then treating the support unit which contains the soluble encoding metal salts with a solution of a salt having an anion that forms insoluble or poorly soluble salts when combined with the encoding metal salts, thereby yielding a stabilized encoding metal salt, which is stabilized against dissolution from the support unit;
wherein step (3) can be carried out before or after step (2) or concurrently with step (2).
The method of preparing an encoded combinatorial library which contains a plurality of ligand-bearing support units, as recited above, wherein the ligands comprise two or more sub-units, comprises the further steps of:
(1) covalently attaching a second sub-unit to the functional group of the first sub-unit, where the second sub-unit may be the same as the first sub-unit or different, where the second sub-unit optionally has a functional group that optionally may be used for adding a third sub-unit;
(2) impregnating the support unit with one or more encoding metal salts, the salts being composed of one or more encoding metal cations, where the encoding metal cations are distributed in their natural isotope abundance or in a non-natural isotope abundance, wherein the encoding metal cations provide a code for identifying the second sub-unit, wherein the encoding metal salts are impregnated onto the support unit by treating the support unit with a solution of a soluble salt of the encoding metal cation to form a support unit labelled with a soluble encoding metal salt, and then treating the support unit labelled with the soluble encoding metal salt with a solution of a salt having an anion that forms insoluble or poorly soluble salts when combined with the encoding metal cations, thereby yielding a stabilized encoding metal salt, which is stabilized against dissolution from the support unit; and
(3) optionally repeating steps (1) and (2) one or more times to add additional sub-units to form a ligand comprising a plurality of sub-units, where the ligand is identifiable by measurement of the distribution of metal cations on the support unit;
wherein step (1) can be carried out before or after step (2) or concurrently with step (2) in each repetition of steps (1) and (2).
A method of preparing an encoded combinatorial library including a plurality of ligand-bearing support units, where the ligand-bearing support units include (a) a solid carrier, (b) one or more ligands covalently bound to the solid carrier, where the ligands are comprised of two or more sub-units, and (c) a plurality of encoding metal salts impregnated on the support unit, the metal salts providing a code for identifying said ligand, comprises the steps of:
(1) providing support units comprising a solid carrier having functional groups, the functional groups being optionally connected to linker groups, the linker groups being organic residues covalently bound to the functional groups of the solid carrier and having functional groups for covalent bonding to the ligand;
(2) covalently attaching a first sub-unit to the functional group of the carrier or to the functional group of the optional linker group (which may consist of more than one molecule bound together, the sub-unit having a functional group for covalent bonding to a second sub-unit;
(3) impregnating the support unit with one or more encoding metal salts, the salts being comprised of one or more encoding metal cations, said encoding metal cations being distributed in their natural isotope abundance or in a non-natural isotope abundance, wherein the combination of encoding metal cations provides a code for identifying the sub-unit that is attached to the support unit, wherein the encoding metal salts are impregnated onto the support unit by treating the support unit with a solution of a soluble salt of the encoding metal cation to form a support unit comprising the soluble encoding metal salt, and then treating the support unit comprising the soluble encoding metal salt with a solution of a salt having an anion that forms insoluble or poorly soluble salts when combined with the encoding metal cations, thereby yielding an encoding metal salt that is stabilized against dissolution from the support unit;
(4) covalently attaching a second sub-unit to the functional group of the first sub-unit, wherein the second sub-unit may be the same as the first sub-unit or different, and the second sub-unit optionally has a functional group that optionally may be used for adding a third sub-unit;
(5) impregnating the support unit with one or more encoding metal salts, the salts being comprised of one or more encoding metal cations, which are distributed in their natural isotope abundance or in a non-natural isotope abundance, where the encoding metal cations provide a code for identifying the second sub-unit; the encoding metal salts are impregnated onto the support unit by treating the support unit with a solution of a soluble salt of the encoding metal cation to form a support unit comprising a soluble encoding metal salt, and then treating the support unit comprising the soluble encoding metal salt with a solution of a salt having an anion that forms insoluble or poorly soluble salts when combined with the encoding metal cations, thereby yielding an encoding metal salt that is stabilized against dissolution from the support unit;
(6) optionally repeating said steps (4) and (5) one or more times to add additional sub-units to form a ligand comprising a plurality of sub-units, where the ligand is identifiable by measurement of the distribution of metal cations on said support unit;
wherein step (2) can be carried out before or after step (3) or concurrently with step (3), step (4) can be carried out before or after step (5) or concurrently with step (5), and in subsequent repetitions of steps (4) and (5) as recited in step (6), step (4) can be carried out before or after step (5) or concurrently with step (5).
In the above description of the method of making the encoded support units, the encoding steps and the steps where sub-units are added are generally part of a split-pool synthesis, which means that the steps described above are carried out on groups of support units rather than individual support units. It also means that after each pair of steps where a ligand or ligand sub-unit is added, along with the addition of a label, the group of support units may be combined with other support units or groups of support units, or the group of support units may be separated into smaller groups, which groups may then be combined with other support units or groups of support units before the next step.
The invention is also an improved method of preparing a combinatorial library comprising a plurality of support units, wherein each support unit comprises (a) a solid carrier, (b) one or more ligands covalently bound to the solid carrier or to a linker group that is covalently bound to the solid carrier, and (c) one or more encoding metal salts impregnated onto the support unit, wherein said encoding metal salts include encoding metal cations, the distribution of the cations providing a code that identifies the ligand or ligands. The improvement is that the encoding metal cations are stabilized against dissolution by treatment of the support units with a solution that comprises one or more anions that form insoluble or poorly soluble salts of said encoding metal cations in the solvent or solvents which are used to prepare said combinatorial library, thereby yielding an encoding metal salt that is stabilized against dissolution.
The combinatorial libraries made by any of the methods described above are also new.
Finally, the method of analyzing the ligands present on the support units in the combinatorial libraries described above utilizes the information encoded in the metal labels. The method of determining the ligand on a single support unit includes the steps of (1) providing a support unit from the library of support units that have ligands and metal labels; (2) analyzing which metal ions are present on the support unit, and (3) comparing the metal ion content with the code information to determine what ligand was synthesized on the support unit.
The metal ion content of the support unit is analyzed using inductively coupled plasma mass spectrometry, and particularly laser ablation inductively coupled plasma mass spectrometry (laser ablation ICP-MS). Laser ablation ICP-MS is an excellent method for carrying out rapid screening of a large number of samples and can be adapted to the screening of an array of samples, such as might be found on a micro-titer plate. An example of an instrument that can be adapted to the analysis of arrays of beads on micro-titer plates is a Perkin Elmer Elan 2000 ICP-MS, coupled with an LSX-200 laser ablation unit from CETAC. Resin beads are plated onto the sample holder and are either scanned with a laser beam or drilled with a laser beam.
The following examples are provided to illustrate the invention. The invention is not to be construed as limited to these specific examples. The scope of the invention is defined by the claims.